A number of NIAID Category A pathogens has developed mechanisms to injure the host through damaging the endothelial barrier to induce progressive vascular leakage. This leads to pulmonary edema and respiratory problems, hemorrhagic fever or shock-like symptoms. We hypothesize that improving vascular resilience and integrity could protect the host against a number of infectious agents. We will investigate the role of key regulators of endothelial integrity using the zebrafish model and in endothelial cell-based assays in the R21 phase of this study. Using anthrax lethal toxin as an inducer of vascular leakage, we will determine how altering host angiogenic signaling could improve endothelial barrier integrity and vascular function. In the R33 phase of this project, we will launch a large-scale chemical library screen, to identify positive hits and characterize their biological effects. We will take advantage of the current knowledge on 3 key angiogenic factors and their receptors, VEGF-A/VEGFR2, Angiopoietin 1/Tie2 and ephrinB2/EphB4, to establish reliable models for vascular integrity. These models will be used to identify small molecules that can improve vascular resilience as potential host-targeted therapies. We propose the following Specific Aims to be completed in the R21 phase: Aim 1. To establish the distinct roles of vascular ligands in comparison with anthrax lethal toxin (LT), in the regulation of permeability using zebrafish and endothelial cell models; Aim 2. To develop conditions for a small scale chemical compound screening using the zebrafish and endothelial cell models of vascular permeability through the use of NERCE chemical resources. Achievement of these Aims will be evaluated through 5 Milestones that would enable progression to the R33 phase of this project. The Aims for the R33 are as follows: Aim 3. To screen chemical libraries for compounds that can improve vascular integrity, R33, Y3-4; Aim 4. To evaluate the dose-response of selected positive compounds and determine whether structural alterations can improve vascular resilience while reducing adverse effects in the zebrafish model, as well as in blood and lymphatic endothelial cell leakage assays, R33, Y4-5. This project is designed to test a hypothesis on vascular protection as a host-targeted approach to pathogen challenge. We envisioned that positive results in the R21 phase will help define the screening strategy in the R33 phase of this project. Access to local chemical resources and expertise will expedite these efforts. PUBLIC HEALTH RELEVANCE: Pathogens induce specific effects while disrupting host physiology. Although activation of the host immune system is an important step, many infectious agents alter cardiovascular function through damage to endothelial cells. When this occurs, the patient suffers from respiratory problems because the lungs are filled with fluid that leaked out o the blood vessels. Endothelial cells line the entire vascular tree in the human body, covering approximately 100 m2 of surface area and providing the largest host tissue for pathogen action. By understanding how to improve vascular integrity and identifying drugs that can induce endothelial resilience, these drugs might be widely protective against a number of biodefense-relevant pathogens.